Compositions, methods and kits for treating cancer

ABSTRACT

Compositions, kits and methods for treating leukemia in a subject (e.g., human) include a first anti-cancer drug consisting of: Δ 12 -prostaglandin J 3  or a derivative thereof, or a prostaglandin D receptor (DP) agonist. The compositions may further include a second anti-cancer drug. Δ 12 -prostaglandin J 3  is a stable metabolite of omega-3 fatty acid, eicosapentaenoic acid (EPA), and was discovered to have anti-leukemic properties. Δ 12 -prostaglandin J 3  was shown to be highly effective in eradicating the leukemia stem cells (LSC) in two murine models of leukemia, thus increasing the survival of the mice. DP agonists were shown to induce apoptosis of human primary Acute Myelogenous Leukemia cells and may be used in compositions, kits and methods for treating leukemia in a subject. The compositions, kits and methods may be particularly useful for treating human subjects who are resistant to one or more anti-cancer drugs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Nonprovisionalapplication Ser. No. 14/335,020, filed Jul. 18, 2014, which is acontinuation application of U.S. Nonprovisional application Ser. No.13/538,297, filed Jun. 29, 2012, now U.S. Pat. No. 8,802,680, issuedAug. 12, 2014, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/502,677, filed Jun. 29, 2011, U.S. Provisional ApplicationSer. No. 61/535,149, filed Sep. 15, 2011, and U.S. ProvisionalApplication Ser. No. 61/635,458, filed Apr. 19, 2012, all of which arehereby incorporated by reference in their entireties, for all purposes,herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Hatch Act ProjectNo. PEN04202, awarded by the United States Department ofAgriculture/NIFA. The Government has certain rights in the invention

FIELD OF THE INVENTION

The invention relates generally to the fields of molecular genetics,molecular biology, and oncology.

BACKGROUND

Leukemia is a highly prevalent disease that signifies uncontrolledproduction of white blood cells. Currently, there is no cure forleukemia. Current therapies of leukemia include chemotherapy, radiationtherapy, stem cell therapy, and biological therapy. All of thesetherapies suffer from many side effects. Use of anti-leukemic drugs onlyprolong the life of the patient by targeting the bulk cancer cells, butnot the cancer stem cells.

SUMMARY

Described herein are compositions, methods and kits for treating cancerssuch as leukemia. Targeting cancer stem cells (CSC) is of paramountimportance to successfully combat the relapse of cancer. It is shownherein that Δ¹²-PGJ₃, a novel and naturally produced cyclopentenoneprostaglandin, CyPG, from the dietary fish-oil omega-3 polyunsaturatedfatty acid (n-3 PUFA), eicosapentaenoic acid (EPA; 20:5), alleviates thedevelopment of leukemia in two well-studied murine models of leukemia.Intraperitoneal administration of Δ¹²-PGJ₃ to mice infected with Frienderythroleukemia virus (FV) or those expressing chronic myelogenousleukemia (CML) oncoprotein BCR-ABL in the hematopoietic stem cell (HSC)pool completely restored normal hematological parameters, splenichistology, and enhanced the survival of such mice. More importantly,Δ¹²-PGJ₃ selectively targeted leukemia stem cells (LSC) for apoptosis inthe spleen and bone marrow. This treatment completely eradicated LSCs invivo as demonstrated by the inability of donor cells from treated miceto cause leukemia in secondary transplants. This is the first example ofa compound that eradicates leukemia stem cells and effectively “cures”CML in a mouse model and prolongs the life of the leukemic miceindefinitely. Given the potency of n-3 PUFA-derived CyPG and thewell-known refractoriness of LSC to currently used clinical agents,Δ¹²-PGJ₃ represents a new chemotherapeutic for leukemia that targetsLSCs.

Accordingly, described herein is a composition including atherapeutically effective amount of a first anti-cancer drug, the firstanti-cancer drug being isolated or synthesized Δ¹²-PGJ₃ or a derivativethereof, for inhibiting LSC growth in a subject having LSCs (e.g., asubject suffering from leukemia) and a pharmaceutically acceptablecarrier. The composition can further include a second anti-cancer drug(e.g., imatinib (Gleevec® Novartis, East Hanover, N.J.)).

Also described herein is a composition including a therapeuticallyeffective amount of a first anti-cancer drug that is an isolated orsynthesized prostaglandin D receptor (DP) agonist for inhibiting LSCgrowth in a subject having LSCs and a pharmaceutically acceptablecarrier. The composition can further include a second anti-cancer drug(e.g., imatinib). The DP agonist can be, for example, one or more of:Δ¹²-PGJ₃, ZK118182, and PGD₂ME.

Further described herein is a method of treating leukemia in a subject.The method includes administering to the subject having leukemia acomposition including a therapeutically effective amount of a firstanti-cancer drug that is one or more of: isolated or synthesizedΔ¹²-PGJ₃ or a derivative thereof, a derivative of prostaglandin D3, andan isolated or synthesized DP agonist, for inducing death of LSCs in thesubject. The LSCs can be, for example, chronic myeloid leukemia stemcells or acute myeloid leukemia cells. In some embodiments, the subjectis resistant to an anti-cancer drug (e.g., imatinib). In the method, thecomposition can further include a therapeutically effective amount of asecond anti-cancer drug (e.g., imatinib, standard chemotherapy agentssuch as cytarabine or doxorubicin, etc.).

Yet further described herein is a method of treating leukemia in asubject (e.g. human). The method includes administering to the subjecthaving leukemia a composition including a therapeutically effectiveamount of a DP agonist for inducing death of LSCs in the subject. TheLSCs can be, for example, chronic myeloid leukemia stem cells. In someembodiments, the subject is resistant to imatinib. In the method, thecomposition can further include a therapeutically effective amount of ananti-cancer drug (e.g., imatinib, standard chemotherapy agents such ascytarabine or doxorubicin, etc.).

Additionally described herein is a kit for treating leukemia in asubject (e.g., human). The kit includes a composition including atherapeutically effective amount of a first anti-cancer drug that is oneof: an isolated or synthesized DP agonist, an isolated or synthesizedΔ¹²-PGJ₃, and a derivative of Δ¹²-PGJ₃, for inducing death of LSCs inthe subject; instructions for use, and packaging. The kit can furtherinclude a second anti-cancer drug (e.g., imatinib, standard chemotherapyagents such as cytarabine or doxorubicin, etc.).

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

By the term “gene” is meant a nucleic acid molecule that codes for aparticular protein, or in certain cases, a functional or structural RNAmolecule.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid).

The terms “patient,” “subject” and “individual” are used interchangeablyherein, and mean a mammalian (e.g., human, rodent, non-human primates,canine, bovine, ovine, equine, feline, etc.) subject to be treatedand/or to obtain a biological sample from.

As used herein, “bind,” “binds,” or “interacts with” means that onemolecule recognizes and adheres to a particular second molecule in asample or organism, but does not substantially recognize or adhere toother structurally unrelated molecules in the sample. Generally, a firstmolecule that “specifically binds” a second molecule has a bindingaffinity greater than about 10⁻⁸ to 10⁻¹² moles/liter for that secondmolecule and involves precise “hand-in-a-glove” docking interactionsthat can be covalent and noncovalent (hydrogen bonding, hydrophobic,ionic, and van der waals).

The term “labeled,” with regard to a probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody.

When referring to a nucleic acid molecule or polypeptide, the term“native” refers to a naturally-occurring (e.g., a wild type or WT)nucleic acid or polypeptide.

As used herein, the term “regulating”, “regulation”, “modulating” or“modulation” refers to the ability of an agent to either inhibit orenhance or maintain activity and/or function of a molecule (e.g., areceptor). For example, an inhibitor of a DP would down-regulate,decrease, reduce, suppress, or inactivate at least partially theactivity and/or function of the DP. Up-regulation refers to a relativeincrease in function and/or activity.

By the term “Δ¹²-PGJ₃” is meant Δ¹²-prostaglandin J₃, an omega-3 fattyacid-derived metabolite.

By the phrase “DP agonist” is meant any agent (e.g., drug, compound,hormone, etc.) that forms a complex with or binds to a DP site on acell, thereby triggering an active response from the cell. DP agonistscan be naturally occurring or synthetic, or a combination thereof.

By the phrase “leukemia stem cells” is meant leukemia initiating cellsthat are functionally defined to possess the property to generate moreleukemia stem cells (self renewal) and non-stem cell leukemia cells.Additionally, these cells are characterized by the expression of certaincell surface markers, which include but are not limited to CD34, CD 123,and CD 117.

The phrases “isolated” or biologically pure” refer to material, which issubstantially or essentially free from components which normallyaccompany it as found in its native state.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled insoluble or bound form, as well as fragments, regions or derivativesthereof, provided by any known technique, such as, but not limited to,enzymatic cleavage, peptide synthesis or recombinant techniques.

As used herein, the terms “diagnostic,” “diagnose” and “diagnosed” meanidentifying the presence or nature of a pathologic condition (e.g.,leukemia).

The term “sample” is used herein in its broadest sense. A sampleincluding polynucleotides, polypeptides, peptides, antibodies and thelike may include a bodily fluid, a soluble fraction of a cellpreparation or media in which cells were grown, genomic DNA, RNA orcDNA, a cell, a tissue, skin, hair and the like. Examples of samplesinclude saliva, serum, blood, urine and plasma.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.Treatment can include, for example, ameliorating, preventing oreliminating splenomegaly, reducing the number of LSCs in a subject,eliminating LSCs in a subject, etc.

As used herein, the term “safe and effective amount” refers to thequantity of a component, which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of acomposition of the present invention effective to yield the desiredtherapeutic response. For example, an amount effective to delay thegrowth of or to cause a cancer (e.g., CML) to shrink or preventmetastasis. The specific safe and effective amount or therapeuticallyeffective amount will vary with such factors as the particular conditionbeing treated, the physical condition of the patient, the type of mammalor animal being treated, the duration of the treatment, the nature ofconcurrent therapy (if any), and the specific formulations employed andthe structure of the compounds or its derivatives.

Although compositions, kits, and methods similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable compositions, kits, and methods are described below.All publications, patent applications, and patents mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Theparticular embodiments discussed below are illustrative only and notintended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E and 1F show endogenous production andpro-apoptotic properties of Δ¹²-PGJ₃. (FIG. 1A) Endogenous formation ofPGD₃, Δ¹²-PGJ₃, 15d-PGJ₃ in RAW264.7 macrophages, LC-UV trace; N=3 forEPA treated. (FIG. 1B) Representative LC-MS of Δ¹²-PGJ₃ containingeluates with characteristic fragmentation pattern is shown. (FIG. 1C)Dose-response demonstrating the effect of Δ¹²-PGJ₃ on BCR-ABL⁺LSCcompared with normal HSCs (MSCV-GFP⁺HSC). Cells were treated ex vivowith Δ¹²-PGJ₃ for 36 h. Apoptosis was measured by annexin V staining.(FIG. 1D) Kit⁺Sca-1⁺Lin⁻BCR-ABL-GFP⁺ cells sorted from the bone marrowand cultured ex vivo in media containing Δ¹²-PGJ₃ (25 nM) or vehiclecontrol for 36 h followed by flow cytometric analysis of GFP⁺ cells.N=3; Mean±s.e.m. shown. *p<0.005. Expressed as percent of input GFP⁺cells. (FIG. 1E) Dose response of LSCs isolated from FV mice withindicated concentrations of Δ¹²-PGJ₃ at the end of 36 h of incubation.Apoptosis of LSCs was examined by Annexin V staining followed by flowcytometry. (FIG. 1F) FV-LSCs were cultured ex vivo with 25 nM of eachcompound for 36 h. N=3; Mean±s.e.m. shown *P<0.0001 (compared to PGJ₃).

FIGS. 2A, 2B, 2C, and 2D show intraperitoneal administration of Δ¹²-PGJ₃eradicates FV-leukemia in mice. (FIG. 2A) Spleen weight of FV-infectedmice treated with various doses of Δ¹²-PGJ₃ (mg/kg body weight). N=10per treatment group. Δ¹²-PGJ₃ treatment at indicated dosage was startedat 1 week post infection for a period of 7 days. *P<0.05. Inset:representative spleens from each treatment group. UI: Uninfected mice.(FIG. 2B) Analysis of LSCs (M34⁺Kit⁺Sca1⁺) in the spleens of FV-infectedmice treated with Δ¹²-PGJ₃ or vehicle (Veh) control. (FIG. 2C) CFU-FVcolony formation in Δ¹²-PGJ₃ and vehicle control treated mice, *P<0.001.(FIG. 2D) H&E staining of spleen sections from uninfected (left),FV-infected-vehicle treated (middle), and FV-infected-Δ¹²-PGJ₃ -treatedmice (right) on day 14 post infection. Small box indicated on eachsection on the left is magnified on the right side. Scale bars, 500 μm.

FIGS. 3A, 3B, 3C, and 3D show the effect of Δ¹²-PGJ₃ treatment onleukemia induced by transplanting FV-induced LSCs expanded in-vitro intoFV-resistant Stk^(−/−) mice. (FIG. 3A) Photograph of spleens fromStk^(−/−) mice seven weeks after transplant with FV-LSCs followed bytreatment with vehicle, 0.05 mg/kg, or 0.025 mg/kg Δ¹²-PGJ₃ for 1 week.(FIG. 3B) Spleen weights are shown for the conditions in panel A. N=5per group, *P<0.05 compared to infected vehicle group. (FIG. 3C) WBCcounts in LSC-transplanted Stk^(−/−) mice treated with indicated amountsof Δ¹²-PGJ₃ or vehicle control. N=5 per group, *P<0.05 compared toinfected vehicle group. (FIG. 3D) M34⁺Kit⁺Sca1⁺ cells in Stk^(−/−) micetransplanted with LSCs. Spleen cells that were isolated and gated onKit⁺, expression of M34 and Sca1 is shown. N=5 per group.

FIGS. 4A, 4B, 4C, 4D, 4E and 4F show that intraperitoneal administrationof Δ¹²-PGJ₃ eradicates LSCs and prolongs survival in a murine CML model.(FIG. 4A) Analysis of the effect of Δ¹²-PGJ₃ treatment on thedevelopment of splenomegaly in mice transplanted with BCR-ABL-GFP⁺LSCs.Representative photographs of spleens from control and BCR-ABLtransplanted mice treated with Δ¹²-PGJ₃ (0.025 mg/kg) or vehicle controlwith corresponding spleen weights. N=10 per treatment group, *P<0.05.(FIG. 4B) Analysis of WBC counts of BCR-ABL⁺LSC or MSCV-HSC transplantedmice treated with Δ¹²-PGJ₃ or vehicle control. *P<0.0001. (FIG. 4C) Flowcytometric analysis of Sca-1⁺Kit⁺GFP⁺cells in the spleen of micetransplanted with BCR-ABL⁺LSC or MSCV⁺HSC treated with Δ¹²-PGJ₃ orvehicle control. N=5 per group; *p<0.001 (FIG. 4D) Analysis of LSCs(Kit⁺Sca-1⁺Lin⁻GFP⁺) in the bone marrow of BCR-ABL⁺LSC transplanted andΔ¹²-PGJ₃-treated mice after 5 weeks of last dose of Δ¹²-PGJ₃ (0.025mg/kg). As a control, BCR-ABL⁺LSC transplanted mice treated with vehiclefor 1 week was used for comparison. (FIG. 4E) Survival curves of micetransplanted with BCR-ABL⁺LSCs or MSCV-GFP⁺HSCs upon treatment withΔ¹²-PGJ₃ (0.025 mg/kg) or vehicle. N=8 per treatment group. (FIG. 4F).HSC were isolated from the bone marrow of C57BL/6 mice and plated inmethylcellulose (1×10⁶ cells/ml/well; Epo, SCF, IL-3, and BMP4) with PBSor Δ¹²-PGJ₃ (25 nM) and cultured for a week. Hematopoietic colonies(colony forming cells in culture, CFC) were scored. Data shown isrepresentative of triplicate experiments.

FIGS. 5A, 5B, 5C, 5D and 5E demonstrate that secondary transplantationof spleen cells from Δ¹²-PGJ₃-treated recipients show absence ofleukemia. Panels A-C represent secondary transplantation ofCD45.1+BCR-ABL mice treated with Δ¹²-PGJ₃ or vehicle controltransplanted into CD45.2 recipient mice. Panels D-E represent FV-LSCsfrom Δ¹²-PGJ₃ or vehicle control treated mice were transplanted intosecondary BALB/c-Stk^(−/−) recipients. (FIG. 5A). Spleen morphology(upper left), spleen weight (lower left) and WBC counts of secondarytransplant mice receiving donor cells from vehicle treated or Δ¹²-PGJ₃treated donor cells (right). (FIG. 5B) Flow cytometry analysis of spleencells from secondary transplants. Cells were gated on GFP⁺ and theexpression of Kit and Scal are shown. (FIG. 5C). Analysis of donorCD45.1 expression in spleen cells. (FIG. 5D) Spleen morphology (upperleft), spleen weight (lower left), and WBC counts of secondarytransplant mice receiving donor cells from vehicle treated or Δ¹²-PGJ₃treated donor cells (right). (FIG. 5E) Flow cytometry analysis of spleencells from secondary transplants. Cells are gated on M34⁺ and theexpression of Kit and Sca1 is shown.

FIGS. 6A, 6B, 6C, 6D and 6E show spontaneous conversion of PGD₃ to PGJ₃,Δ¹²-PGJ₃, and 15d-PGJ₃ in-vitro.

FIGS. 7A, 7B and 7C show a dose-dependent pro-apoptotic effect of CyPGson LSCs.

FIG. 8 is a graph showing that imatinib-resistant BCR-ABL(GFP)+ cellsare targeted by Δ¹²-PGJ₃. The LSCs were isolated from mice treated withimatinib (75 mg/kg) for one week following which the treatment wasstopped. The mice were followed for the development of leukemia. Micethat developed leukemia were euthanized and spleens were used as thesource of LSCs.

FIG. 9 is a graph showing apoptosis of BCR-ABL⁻ LSCs by syntheticagonists of the DPs.

FIG. 10 is a graph showing that Δ¹²-PGJ₃ and related agonists do notaffect normal human hematopoiesis as measured by the ability of bonemarrow cells to form differentiated colonies when cultured in vitro.Human unfractionated bone marrow cells (5×10⁵ per well) were plated inmethylcellulose complete media containing IL-3, GM-CSF, G-CSF, SCF andEpo supplemented with the indicated concentrations of drugs. Totalcolonies were counted after 12 days.

FIG. 11 is a graph showing that Δ¹²-PGJ₃ does not affect the ability ofnormal bone marrow cells to differentiate in to cells of the erythroidlineage (Burst Forming Units—erythroid, BFU-E).

FIG. 12 is a pair of graphs showing that DP mediate theΔ¹²-PGJ₃-dependent apoptosis of blast crisis CML cells from a patient(#011711).

FIGS. 13A and 13B show results from an experiment in which DP mediatethe Δ¹²-PGJ₃-dependent apoptosis of AML cells from a patient (#100810).Furthermore, Δ₁₂-PGJ₃ also specifically targeted Leukemia stem cells(CD34+CD38−CD123+cells) for apoptosis.

FIG. 14 is a graph showing results from a comparison of Δ¹²-PGJ₃ withImatinib (Gleevec® Novartis, East Hanover, N.J.) in the BCR-ABL⁺ LSCtransplant CML model in mice.

FIG. 15 is a Table listing the effect of Δ¹²-PGJ₃ on LSCs from AML andblast-crisis CML patients.

FIG. 16 is a pair of graphs showing apoptosis of human primary AML cellsby DP agonists (endogenous and exogenous) and DP antagonists.

DETAILED DESCRIPTION

AML is one of the most common types of leukemia in adults.Unfortunately, the five year relative survival rates for AML are thelowest when compared to other forms of leukemia. AML is a stem celldisease where LSCs occupy the apex of the disease hierarchy. LSCs canself renew and generate non-stem cell progeny that make up the bulk ofthe leukemia cells. Although chemotherapy agents can effectively targetbulk leukemia cells, LSCs have active mechanisms to avoid killing bythese drugs. As a consequence, failure to eliminate LSCs results inrelapse of the disease. Because of this property, specific targeting ofLSCs is essential for successful treatment. Although the need for newanti-LSC based therapies is well recognized, the identification ofmechanism-based drugs to target LSCs has been lacking. Clearly newapproaches are needed. Described herein are compositions, methods andkits for treating cancer (e.g., leukemia). A metabolite derived from ω-3fatty acids, Δ¹²-PGJ₃, was discovered which effectively eradicates LSCsin two mouse models of chronic leukemia. In the experiments describedherein, these findings were extended to show that Δ¹²-PGJ₃ effectivelytargets AML LSCs by inducing apoptosis in murine models of AML and inhuman AML leukemia samples. In contrast, PGJ₃ has no effect on normalhematopoietic stem cells or the differentiation of hematopoieticprogenitors. Δ¹²-PGJ₃ acts by inducing the expression of p53 in LSCs andleukemia cells. High-level expression of p53 in LSCs is incompatiblewith self renewal and leads to apoptosis. These data suggest thatΔ¹²-PGJ₃ is a chemotherapeutic agent for treating AML. This is the firstexample of a compound that eradicates leukemia stem cells andeffectively “cures” CML in a mouse model and prolongs the life of theleukemic mice indefinitely.

Biological Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).

Compositions for Treating Leukemia in a Subject

Described herein are compositions for treating leukemia in a subject(e.g., a human subject). Examples of leukemias that can be treated usingthe compositions include Acute Myelogenous Leukemia (AML), CML, AcuteLymphocytic Leukemia (ALL) and Chronic Lymphocytic Leukemia (CLL). Inone embodiment, a composition includes a therapeutically effectiveamount of Δ¹²-prostaglandin J₃, or a derivative thereof (a firstanti-cancer drug), for inhibiting LSC growth in a subject having LSCs,and a pharmaceutically acceptable carrier. Inhibiting LSC growthincludes inducing death (killing of) of the cancer cells, and/orinducing differentiation of the cancer cells (promoting a moredifferentiated phenotype, e.g., causing differentiation of LSCs intoterminally differentiated cells). Any suitable form of Δ¹²-prostaglandinJ₃ or derivative thereof can be used (e.g., synthesized, isolated).Δ¹²-prostaglandin J₃ derivatives that may find particular use in thecompositions and methods described herein are those that induceapoptosis or differentiation of LSCs (e.g., 16,16-dimethyl-Δ¹²-PGJ₃). Insuch embodiments, when administered to a subject, the compositioninduces apoptosis of LSCs. The composition can further include one ormore additional anti-cancer drugs (e.g., a second anti-cancer drug).Examples of additional anti-cancer drugs include imatinib, nilotinib,dasatinib, new generation BCR-ABL inhibitors, and standard chemotherapydrugs such as cytarabine or doxorubicin or similar classes of drugs. Inone embodiment, a combination therapy including imatinib or a newgeneration BCR-ABL inhibitor and Δ¹²-PGJ₃ may be particularlytherapeutic.

In another embodiment, a composition includes a therapeuticallyeffective amount of a DP agonist (a first anti-cancer drug) forinhibiting LSC growth in a subject having LSCs and a pharmaceuticallyacceptable carrier. Examples of DP agonists include a small molecule, aprotein, a peptide, a polynucleotide, an oligonucleotide, an organiccompound, an inorganic compound, synthetic compounds or compoundsisolated from unicellular or multicellular organisms. Specific examplesof DP agonists include PGD₂ME (Prostaglandin D₂ methyl ester(9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid, methyl ester)and ZK118182 ([[4-[5R-chloro-2Z-[3R-cyclohexyl-3S-hydroxy-1R-propenyl]-3S-hydroxycyclopentyl]-2R-butenyl]oxy]-acetic acid, isopropylester). An agonist of a DP is any agent that activates the DP. Any agentthat activates DP can be used in compositions and methods describedherein for inducing death of LSCs and treating leukemia. A compositionincluding a DP agonist can further include one or more additionalanti-cancer drugs (e.g, a second anti-cancer drug). As noted above,examples of additional anti-cancer drugs include imatinib, nilotinib,dasatinib, new generation BCR-ABL inhibitors, standard chemotherapydrugs such as cytarabine or doxorubicin, etc.

In the compositions described herein, Δ¹²-prostaglandin J₃ can beobtained commercially or synthesized according to the methods described,for example, in the Examples section below. Similarly, Δ¹²-prostaglandinJ₃ derivatives can be synthesized as described by Kimball et al.(Kimball F A, Bundy G L, Robert A, and Weeks J R (1979), Synthesis andbiological properties of 9-deoxo-16,16-9-methylene-PGE2. Prostaglandins17: 657-66).

Effective Doses

The compositions described above are preferably administered to a mammal(e.g., rodent, human, non-human primates, canine, bovine, ovine, equine,feline, etc.) in an effective amount, that is, an amount capable ofproducing a desirable result in a treated subject (e.g., inhibitinggrowth of LSCs and/or inducing death of LSCs in the subject). Toxicityand therapeutic efficacy of the compositions utilized in methods of theinvention can be determined by standard pharmaceutical procedures. As iswell known in the medical and veterinary arts, dosage for any one animaldepends on many factors, including the subject's size, body surfacearea, body weight, age, the particular composition to be administered,time and route of administration, general health, the clinical symptomsof the cancer and other drugs being administered concurrently. Acomposition as described herein is typically administered at a dosagethat induces death of LSCs (e.g., induces apoptosis of LSCs), as assayedby identifying a reduction in hematological parameters (Complete bloodcount (CBC)), or cancer cell growth or proliferation. In the experimentsdescribed herein, the amount of Δ¹²-PGJ₃ used to eradicate LSCs wascalculated to be 0.6 micrograms/day/gram mouse for 7 days. Generally,the dose is in mg/Kg subject/day=ug/g subject/day. In a typicalembodiment, a dose in the range of about 0.025 to about 0.05 mg/Kg/dayis administered. Such a dose is typically administered once a day for afew weeks.

Methods of Treating Cancer

Described herein are methods of treating cancer (e.g., leukemia) and/ordisorders or symptoms thereof. The methods include administering atherapeutically effective amount of a pharmaceutical compositionincluding a pharmaceutically acceptable carrier and an amount ofΔ¹²-PGJ₃, a derivative thereof, or a DP agonist (a first anti-cancerdrug) sufficient to treat the disease or disorder or symptom thereof toa subject (e.g., a mammal such as a human). In the method, an amount ofΔ¹²-PGJ₃, a derivative thereof, or a DP agonist sufficient to inducedeath of LSCs in the subject is typically administered. In a typicalembodiment, the LSCs are CML stem cells. In some embodiments, thecomposition can be administered to a subject who is resistant toimatinib or other anti-cancer drug. In the methods, the composition canfurther include a therapeutically effective amount of one or moreadditional anti-cancer drugs (e.g., a second anti-cancer drug such asimatinib) or standard chemotherapy.

The therapeutic methods of the invention (which include prophylactictreatment) in general include administration of a therapeuticallyeffective amount of the compositions described herein to a subject inneed thereof, including a mammal, particularly a human. Such treatmentwill be suitably administered to subjects, particularly humans,suffering from, having, susceptible to, or at risk for a disease,disorder, or symptom thereof. Determination of those subjects “at risk”can be made by any objective or subjective determination by a diagnostictest or opinion of a subject or health care provider (e.g., genetictest, enzyme or protein marker, marker (as defined herein), familyhistory, and the like).

The administration of a composition including Δ¹²-PGJ₃, a derivativethereof, or a DP agonist for the treatment of cancer (e.g., leukemia)may be by any suitable means that results in a concentration of thetherapeutic that, (e.g., when combined with other components), iseffective in ameliorating, reducing, or stabilizing a cancer. TheΔ¹²-PGJ₃, a derivative thereof, or a DP agonist may be contained in anyappropriate amount in any suitable carrier substance, and is generallypresent in an amount of 1-95% by weight of the total weight of thecomposition. The composition may be provided in a dosage form that issuitable for local or systemic administration (e.g., parenteral,subcutaneously, intravenously, intramuscularly, or intraperitoneally).The pharmaceutical compositions may be formulated according toconventional pharmaceutical practice (see, e.g., Remington: The Scienceand Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, LippincottWilliams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology,eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).

Compositions as described herein may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non-toxic pharmaceutically acceptable carriers andadjuvants. The formulation and preparation of such compositions are wellknown to those skilled in the art of pharmaceutical formulation.Formulations can be found in Remington: The Science and Practice ofPharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms(e.g., in single-dose ampoules), or in vials containing several dosesand in which a suitable preservative may be added (see below). Thecomposition may be in the form of a solution, a suspension, an emulsion,an infusion device, or a delivery device for implantation, or it may bepresented as a dry powder to be reconstituted with water or anothersuitable vehicle before use. Apart from the active agent that reduces orameliorates a cancer, the composition may include suitable parenterallyacceptable carriers and/or excipients. The active therapeutic agent(s)may be incorporated into microspheres, microcapsules, nanoparticles,liposomes, or the like for controlled release. Furthermore, thecomposition may include suspending, solubilizing, stabilizing,pH-adjusting agents, tonicity adjusting agents, and/or dispersingagents.

As indicated above, the pharmaceutical compositions described herein maybe in a form suitable for sterile injection. To prepare such acomposition, the suitable active therapeutic(s) are dissolved orsuspended in a parenterally acceptable liquid vehicle. Among acceptablevehicles and solvents that may be employed are water, water adjusted toa suitable pH by addition of an appropriate amount of hydrochloric acid,sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer'ssolution, and isotonic sodium chloride solution and dextrose solution.The aqueous formulation may also contain one or more preservatives(e.g., methyl, ethyl or n-propyl p-hydroxybenzoate). In cases where oneof the compounds is only sparingly or slightly soluble in water, adissolution enhancing or solubilizing agent can be added, or the solventmay include 10-60% w/w of propylene glycol or the like.

Materials for use in the preparation of microspheres and/ormicrocapsules are, e.g., biodegradable/bioerodible polymers such aspolygalactin, poly-(isobutyl cyanoacrylate),poly(2-hydroxyethyl-L-glutam-nine) and, poly(lactic acid). Biocompatiblecarriers that may be used when formulating a controlled releaseparenteral formulation are carbohydrates (e.g., dextrans), proteins(e.g., albumin), lipoproteins, or antibodies. Materials for use inimplants can be non-biodegradable (e.g., polydimethyl siloxane) orbiodegradable (e.g., poly(caprolactone), poly(lactic acid),poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Formulations for oral use include tablets containing the activeingredient(s) (e.g., Δ¹²-PGJ₃ or a derivative thereof, a DP agonist) ina mixture with non-toxic pharmaceutically acceptable excipients. Suchformulations are known to the skilled artisan. Excipients may be, forexample, inert diluents or fillers (e.g., sucrose, sorbitol, sugar,mannitol, microcrystalline cellulose, starches including potato starch,calcium carbonate, sodium chloride, lactose, calcium phosphate, calciumsulfate, or sodium phosphate); granulating and disintegrating agents(e.g., cellulose derivatives including microcrystalline cellulose,starches including potato starch, croscarmellose sodium, alginates, oralginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia,alginic acid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

The tablets may be uncoated or they may be coated by known techniques,optionally to delay disintegration and absorption in thegastrointestinal tract and thereby providing a sustained action over alonger period. The coating may be adapted to release the active drug ina predetermined pattern (e.g., in order to achieve a controlled releaseformulation) or it may be adapted not to release the active drug untilafter passage of the stomach (enteric coating). The coating may be asugar coating, a film coating (e.g., based on hydroxypropylmethylcellulose, methylcellulose, methyl hydroxyethylcellulose,hydroxypropylcellulose, carboxymethylcellulose, acrylate copolymers,polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating(e.g., based on methacrylic acid copolymer, cellulose acetate phthalate,hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcelluloseacetate succinate, polyvinyl acetate phthalate, shellac, and/orethylcellulose). Furthermore, a time delay material, such as, e.g.,glyceryl monostearate or glyceryl distearate may be employed.

Optionally, a composition as described herein may be administered incombination with any other anti-cancer therapy (e.g., imatinib); suchmethods are known to the skilled artisan and described in Remington: TheScience and Practice of Pharmacy, supra. In one example, an effectiveamount of Δ¹²-PGJ₃, a derivative thereof, or a DP agonist isadministered in combination with radiation therapy. Combinations areexpected to be advantageously synergistic. Therapeutic combinations thatinhibit cancer (e.g., leukemia) cell growth and/or induce apoptosis ofLSCs are identified as useful in the methods described herein.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof changes in hematological parameters and LSC analysis with cellsurface proteins as diagnostic markers (which can include, for example,but are not limited to CD34, CD38, CD90, and CD117) or diagnosticmeasurement (e.g., screen, assay) in a subject suffering from orsusceptible to a disorder or symptoms thereof associated with cancer(e.g., leukemia) in which the subject has been administered atherapeutic amount of a composition as described herein. The level ofmarker determined in the method can be compared to known levels ofmarker in either healthy normal controls or in other afflicted patientsto establish the subject's disease status. In preferred embodiments, asecond level of marker in the subject is determined at a time pointlater than the determination of the first level, and the two levels arecompared to monitor the course of disease or the efficacy of thetherapy. In certain preferred embodiments, a pre-treatment level ofmarker in the subject is determined prior to beginning treatmentaccording to the methods described herein; this pre-treatment level ofmarker can then be compared to the level of marker in the subject afterthe treatment commences, to determine the efficacy of the treatment.

Kits for Treating Leukemia in a Subject

Described herein are kits for treating leukemia in a subject. A typicalkit includes a composition including a therapeutically effective amountof a DP agonist or of Δ12-prostaglandin J3 or a derivative thereof (afirst anti-cancer drug) for inducing death of LSCs in a subject,packaging, and instructions for use. In a kit, the composition mayfurther include a pharmaceutically acceptable carrier in unit dosageform. If desired, the kit also contains an effective amount of anadditional anti-cancer drug (e.g., a second anti-cancer drug such asimatinib). In some embodiments, the kit includes a sterile containerwhich contains a therapeutic or prophylactic composition; suchcontainers can be boxes, ampules, bottles, vials, tubes, bags, pouches,blister-packs, or other suitable container forms known in the art. Suchcontainers can be made of plastic, glass, laminated paper, metal foil,or other materials suitable for holding medicaments.

EXAMPLES

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the invention in any way.

Example 1 Δ¹²-Prostaglandin J₃, an Omega-3 Fatty Acid-DerivedMetabolite, Selectively Ablates LSCs in Mice

The endogenous formation of Δ¹²-PGJ₃ from EPA was investigated and theability of this novel n-3 PUFA metabolite to target LSCs was examined intwo well-studied models of leukemia, Friend Virus (FV)-inducederythroleukemia (Ben-David Y & Bernstein A, Cell. 1991;66:831-834) and awell-established model for inducing CML in mice, which utilizesBCR-ABL-IRESGFP retrovirus (Schemionek M et al., Blood.2010;115:3185-3195; Pear W S et al., Blood. 1998;92:3780-3792; Hu Y etal., Proc Natl Acad Sci USA. 2006;103:16870-16875; Zhao C et al.,Nature. 2009;458:776-779), wherein transplantation of transduced HSCsinto mice results in pathology similar to the chronic phase of CML.FV-induces leukemia by activating the bone morphogenetic protein-4(BMP4)-dependent stress erythropoiesis pathway, which leads to a rapidamplification of target cells and acute disease (Subramanian A et al., JVirol. 2008;82:382-393). The results described herein demonstrate thatΔ¹²-PGJ₃ administration (at doses as low as 0.6 μg/g mouse/day) toFV-infected and BCR-ABL⁺ transduced HSC (hereafter referred to asBCR-ABL⁺LSC) transplanted mice completely ablates leukemia, restores thehematological parameters, and eradicates LSC via the activation ofATM/p53 pathway of apoptosis in these cells.

METHODS

Cell culture. Murine erythroleukemia (MEL) cells were cultured in DMEMwith 10% FBS. In order to examine the production 3-series PGs,BALB/c-derived RAW264.7 macrophage-like cells (ATCC) were cultured inDMEM containing 5% FBS, 250 nM sodium selenite, and 50 μM EPA (as BSAconjugate) for 72 h followed by stimulation with E. coli endotoxinlipopolysaccharide (LPS; Serotype 0111B4; 50 ng/ml) for 30 min. Thecells were cultured in fresh DMEM for an additional 24-144 h. Cellscultured with cell culture-grade fatty acid free BSA (Sigma Aldrich)served as a control. Culture media was withdrawn at various times andanalyzed for 3-series PGs as described below. Total RNA was isolatedfrom cells or tissues using Trizol reagent as per the instructions ofthe supplier (Invitrogen, Carlsbad, Calif.) and cDNA was prepared usinga High Capacity cDNA Reverse Transcriptase kit (Applied Biosystems,Foster City, Calif.). Semiquantitative RT-PCR for p53 and β-actin wasperformed with primers as described in Supplementary Methods. Nuclearand cytoplasmic protein extracts of LSCs were prepared using standardmethods previously described (Vunta H et al., J Biol Chem.2007;282:17964-17973).

Preparation, isolation, and spectroscopic characterization of PGD₃metabolites. PGD₃ (Cayman Chemicals) was incubated with 0.1 M sodiumphosphate buffer, pH 7.4, containing 0.9% NaCl at a final concentrationof 100 μg/ml with shaking at 37° C.. for varying periods (24 h-144 h).The reaction products and those from the cell culture media supernatantswere purified by HPLC and analyzed by UV and MS as described below inSupplementary Information and Methods.

Apoptosis. Apoptosis of LSCs was performed using annexin V as describedbelow in Supplementary Information and Methods.

FV-induced erythroleukemia and production of FV leukemia stem cells(FV-LSCs): BALB/c mice were infected with FV as previously described(Subramanian A et al., J Virol. 2008;82:382-393; Harandi OF et al., JClin Invest. 2010;120:4507-4519). On day 14 after infection, spleenswere isolated and a single cell suspension of spleen cells wasgenerated. The cells were filtered through a 70 μm sterile filter andflow-through cells were resuspended in RBC lysis buffer followed bycentrifugation. Leukemia stem cells were isolated by FACS. Spleen cellswere labeled with anti-Kit, Scal (BioLegend, San Diego, Calif.) and M34antibodies. M34 is a monoclonal antibody that recognizes the envelopeprotein of SFFV (Chesebro B et al., Virology. 1981;112:131-144) and wasused as previously described (Subramanian A et al., J Virol.2008;82:382-393). As indicated M34⁺Kit⁺Sca1⁺ cells were cultured inMethocult media (Stem Cell Technologies Vancouver BC) M3334 supplementedwith 200 ng/ml Sonic Hedgehog (Shh), 15 ng/ml bone morphogeneticprotein-4 (BMP4) (both from R&D Systems Minneapolis, Minn.), and 50ng/ml stem cell factor (SCF; Peprotech). For CFU-FV assays, cells wereplated in methylcellulose media containing fetal calf serum, but lackingadded growth factors as previously described (Mager DL et al., Proc NatlAcad Sci USA. 1981;78:1703-1707).

Transplant of FV-LSCs into BALB/c-Stk^(−/−) mice: FV-LSCs were generatedas described above. 2.5×10⁵ FV-LSCs were transplanted intoBALB/c-Stk^(−/−) mice by retro-orbital injection. Six weeks aftertransplant the mice were treated with CyPGs or vehicle control asindicated in the text.

Induction of CML using MIGR-BCR-ABL retrovirus: MIGR-BCR-ABL and controlMSCV-GFP retroviruses were obtained. Viral stocks were generated inHEK293 cells as previously described (Finkelstein L et al., Oncogene.2002;21:3562-3570). C57BL/6 mice were treated with 5-fluorouracil (5-FU;150 mg/Kg, Sigma, St. Louis, Mo.) to enrich for cycling HSCs. On dayfour after treatment bone marrow cells were harvested and infected withMIGR-BCR-ABL or MSCV-GFP control virus overnight in IMDM mediacontaining 5% FCS and supplemented with 2.5 ng/ml IL-3 and 15 ng/ml SCF(R&D Systems Minneapolis, Minn.). 0.5×10⁶ transduced cells weretransplanted by retro-orbital injection into C57BL/6 recipient mice thatwere preconditioned with 950 Rads of irradiation. In order to increasethe number of CML and control mice, 17 days after transplant GFP⁺ spleencells were isolated by FACS and 1×10⁵ GFP⁺ cells were transplanted intoirradiated (950 Rads) secondary C57BL/6 recipients. Two weeks aftertransplant, mice were treated as indicated with CyPGs and vehiclecontrol. For ex-vivo experiments, Kit⁺Sca1⁺Lin⁻GFP⁺ cells were isolatedfrom the bone marrow or spleen of transplanted mice by FACS. The sortedcells were cultured in Methocult media M3334 (Stem Cell TechnologiesVancouver BC) M3334 supplemented with Shh, SCF, and BMP4 and treatedwith the indicated CyPGs and vehicle controls for indicated timeperiods. To demonstrate the effect of Δ¹²-PGJ₃ on normal hematopoieticprogenitors, HSCs isolated from the bone marrow of C57BL/6 mice werecultured in methylcellulose media (1×10⁶ cells/ml/well) containing Epo(3 U/ml), SCF, IL-3, and BMP4 in the presence or absence of Δ¹²-PGJ₃ (25nM). The hematopoietic colonies (colony forming cells in culture, CFC)were scored.

Secondary transplants to test for residual LSCs after treatment withΔ¹²-PGJ₃: For the CML model, B6.SJLPtprca Pep3b/BoyJ (CD45.1⁺) mice weretreated with 5-FU and bone marrow cells enriched in cycling HSCs wereisolated followed by infection with MIGR-BCR-ABL virus or controlMSCV-GFP virus as described above. The cells were transplanted intoC57BL/6 (CD45.2) recipient mice as mentioned earlier. The mice weretreated with Δ¹²-PGJ₃ or vehicle control as indicated. Two weeks aftertreatment, spleen cells were isolated and transplanted into irradiatedsecondary C57BL/6 (CD45.2) recipients as described above. Two weeksafter secondary transplant, mice were analyzed for WBC counts,splenomegaly and the presence of GFP⁺ or CD45.1 donor cells in the bonemarrow and spleen by flow cytometry. Secondary transplants were alsodone with FV infected mice treated with Δ¹²-PGJ₃ or vehicle control.BALB/c mice were infected with Friend virus as described above. The micewere treated with Δ¹²-PGJ₃ or vehicle control as indicated. Two weeksafter treatment, spleen cells isolated from FV-infected mice andtransplanted into BALB/c-Stk^(−/−) recipient mice (1×10⁵ cells permouse). Five weeks post transplant, the mice with secondary transplantswere tested for WBC counts, splenomegaly and for the presence ofM34⁺Kit⁺Sca1⁺FV-LSCs by flow cytometry.

Treatment of Mice with PGs: Mice with FV-induced erythroleukemia orMIGR-BCR-ABL induced CML were treated on the indicated days with CyPGs.Mice were treated with a daily intraperitoneal injection of Δ¹²-PGJ₃(0.01-0.1 mg/kg), 15d-PGJ₂ (0.1 mg/kg), or 9,10-dihydro-15d-PGJ₂ (0.1mg/kg) for 7 days. All three compounds were formulated withhydroxypropyl-β-cyclodextrin (30% w/v; Sigma; vehicle control). Allexperiments utilizing mice were approved by the IACUC of thePennsylvania State University.

Inhibition of ATM kinase in LSC. LSCs isolated from FV-infected mice orBCR-ABL⁺LSCs transplanted mice were treated with indicatedconcentrations of either ATM-specific inhibitor (MTPO,2-Morpholin-4-yl-6-thianthren-1-yl-pyran-4-one; KU55933; 50 nM;Calbiochem) or ATM/ATR-specific inhibitor (CGK-733; 1 μM; Calbiochem)followed by treatment with CyPGs.

Statistical analysis. The results are expressed as means±s.e.m. and thedifferences between groups were analyzed using Student's t test usingGraphPad Prism. The criterion for statistical significance was P<0.05.

RESULTS

Endogenous metabolites of EPA: To relate the potent antileukemic effectsof EPA-derived CyPGs to their endogenous production, the cellularbiosynthesis of PGD₃, Δ¹²-PGJ₃ and 15d-PGJ₃ was examined in murinemacrophage-like cells (RAW264.7) cultured with EPA (50 μM). RAW264.7cells, which express H-PGDS²⁶, were stimulated with bacterial endotoxinlipopolysaccharide (LPS; 50 ng/ml) to induce expression of COX-2.Treated cells produced detectable amounts of PGD₃ and its metabolites at48 h post-LPS treatment. LC-MS analysis of culture media supernatantsconfirmed the increased production of PGD₃, Δ¹²-PGJ₃, and 15d-PGJ₃ (FIG.1A; FIG. 6) only in cells treated with EPA. Based on the LC-retentiontimes and mass fragmentation patterns, the cellular metabolites wereidentified as PGD₃ (m/z 349; FIG. 6A), Δ¹²-PGJ₃ (m/z 331.45; FIG. 1B)and 15d-PGJ₃ (m/z 313.45; FIG. S1A). These metabolites were not seen incells cultured without exogenous EPA (FIG. 1A). It was estimated thattreatment of macrophages with 50 μM EPA produced ˜0.15 μM ofΔ¹²-PGJ₃/10⁶ cells in 48 h. Non-enzymatic dehydration of PGD₃ inphosphate buffered saline produced PGJ₃, Δ¹²-PGJ₃, and 15d-PGJ₃in-vitro. Incubation of PGD₃ (100 μg/ml; Cayman Chemicals) in a serumfree environment for 24-48 h at 37° C. led to the formation of Δ¹²-PGJ₃,PGJ₃ (Δ¹³-PGJ₃) (PGJ3 is also called D13-PGJ3 due to the unsaturation atcarbon 13; this is a isomer that is formed), and 15d-PGJ₃ that were wellresolved on a reverse phase LC (C₁₈) column with retention times 9.63,9.97, and 11.02 min, respectively (FIGS. 6B, C). Prolonged incubation ofPGD₃ up to 144 h at 37° C. also produced these metabolites, withΔ¹²-PGJ₃ predominating over the others (FIG. 6C). Presence of serum(10%) did not affect the conversion of PGD₃ (FIG. 6E). UV-spectroscopicanalysis of the purified Δ¹²-PGJ₃ confirmed the presence of a conjugateddiene-like structure with a λ_(max) of 242 nm; while PGJ₃ and 15d-PGJ₃showed a distinct peak at ˜300 nm, which is characteristic of thecyclopentenone structure. Together, these data confirm the endogenousproduction of PGD₃ metabolites and the enhanced stability of Δ¹²-PGJ₃ inan aqueous environment. Δ¹²-PGJ₃ induces apoptosis of LSCs: Here, thepro-apoptotic properties of PGD₃ metabolites were examined in the twowell-studied murine models of leukemia. Mice were transplanted with HSCstransduced with a BCR-ABL expressing retrovirus (hereafter referred toas BCR-ABL mice). Incubation of Kit⁺Scal⁺Lin⁻GFP⁺ LSCs isolated from thespleen of BCR-ABL mice with low doses of Δ¹²-PGJ₃ significantlyincreased apoptosis of these cells with an IC₅₀ of ˜12 nM, but did notaffect the normal HSCs, that are represented by Kit⁺Sca1⁺Lin⁻GFP⁺ cellsisolated from mice transplanted with HSCs transduced with a MSCV-GFPcontrol virus (hereafter referred to as MSCV-control mice) (FIG. 1C).Similar effects were also observed when BCR-ABL LSCs (Kit⁺Scal⁺Lin⁻GFP⁺)isolated from the bone marrow were treated ex vivo with PGJ₃ (FIG. 1D).An identical effect was also observed with FV-LSCs (FIG. 1E). Incubationof FV-LSCs with EPA had no effect, while PGJ₃ displayed only a 2-foldincrease in apoptosis, Δ¹²-PGJ₃ and 15d-PGJ₃ treatment at 25 nM led to asignificant increase (˜75%) in apoptosis (FIG. 1F). The effects ofARA-derived PGJ₂, Δ¹²-PGJ₂ and 15d-PGJ₂ on FV-LSCs and LSCs derived fromBCR-ABL mice were also examined. Responses similar to Δ¹²-PGJ₃ withΔ¹²-PGJ₂ and 15d-PGJ₂ were observed, while PGJ₂ was largely ineffective(FIG. 7A). In contrast, there was no apoptosis of FV-LSC treated with9,10-dihydro-15d-PGJ₂, a 15d-PGJ₂ derivative that lacks an unsaturationat carbon-9 (FIG. 7B). Ex vivo treatment of Sca1⁺GFP⁺Kit⁺BCR-ABL⁺LSCsorted from the spleen of transplanted mice with 25-1000 nM of Δ¹²-PGJ₃or 15d-PGJ₂ significantly increased their apoptosis; while9,10-dihydro-15d-PGJ₂ was ineffective even at high concentrations up to1 μM (FIG. 7C). While all the data described herein clearly demonstratedthe proapoptotic ability of Δ¹²-PGJ₃, it was next examined if Δ¹²-PGJ₃modulated NF-κB or PPARγ, which has been shown to be the mechanism bywhich 15d-PGJ₂ induces apoptosis (Rossi et al., Nature.2000;403:103-108; Forman BM et al., Cell. 1995;83:803-812). Δ¹²-PGJ₃ didnot affect the NF-κB pathway as seen by gel shift analysis atconcentrations in high nM range in LPS-treated RAW264.7 cells.Furthermore, analysis of the NF-κB activation in sorted BCR-ABL⁺LSCstreated with Δ¹²-PGJ₃ by EMSA and Western blotting of nuclear extractsalso demonstrated lack of activation of NF-κB. Also, Δ¹²-PGJ₃ was unableto activate PPARγ in reporter assays at nanomolar concentrations thatcaused apoptosis of LSC. Along the same lines, treatment of FV-LSCs withrosiglitazone, a synthetic agonist of PPARγ (Nolte RT et al., Nature.1998;395:137-143) did not affect proliferation of LSC indicating thatthe apoptotic pathway did not involve PPARy (FIG. 7B, inset). Takentogether, the data indicates that an alkylidenecyclopentenone structurein CyPGs is absolutely essential to effectively induce apoptosis of LSCsfrom two murine models of leukemia by a mechanism that does not involvePPARy or NF-κB.

Δ¹²-PGJ₃ eradicates leukemia and alleviates splenomegaly in theFV-infected mice. Given the potent proapoptotic potential of Δ¹²-PGJ₃ onLSC in vitro, the ability of Δ¹²-PGJ₃ to ablate LSCs in FV-infectedleukemic mice was tested. Seven days post infection with FV, the micewere treated with Δ¹²-PGJ₃ at 0.01and 0.05 mg/kg/day for an additionalweek and the mice were euthanized on day 14-post infection. Compared tothe vehicle treated mice, FV-infected mice treated with Δ¹²-PGJ₃ at 0.05(FIG. 2A) and 0.1 mg/kg showed no signs of splenomegaly. Although 0.01mg/kg treatment did not completely ablate splenomegaly, there was asignificant reduction (˜50%) (FIG. 2A). A similar trend was also seenwith 15d-PGJ₂; while 9,10-dihydro-15d-PGJ₂ did not have any effect onthe amelioration of splenomegaly. Flow cytometric analysis clearlydemonstrated that Δ¹²-PGJ₃ (0.05 mg/kg) completely eradicated theScal⁺Kit⁺M34⁺Ter119^(lo) cells in the spleen (FIG. 2B), which representsthe LSC population. Identical results were obtained with 15d-PGJ₂; while9,10-dihydro-15d-PGJ₂-treatment was ineffective. In agreement with theabsence of splenomegaly and complete ablation of LSCs, total leukocyteand reticulocyte counts were decreased to normal levels in Δ¹²-PGJ₃ aswell as in 15d-PGJ₂-treated mice. Previous work has shown thattransformed leukemia cells form colony forming units-Friend virus(CFU-FV) that exhibit factor-independent growth, which can be measuredby plating infected spleen cells in methylcellulose media without growthfactors (Mager D L et al., Proc Natl Acad Sci USA. 1981;78:1703-1707).CFU-FV in the Δ¹²-PGJ₃-treated mice was completely reduced to backgroundlevels, similar to those in the uninfected mice (FIG. 2C). Histologicalexamination of the vehicle-treated FV-infected spleen showed completeeffacement of splenic architecture as a result of infiltration ofleukemic blasts, with erythroid progenitor expansion replacing thesinusoids (FIG. 2D). Consistent with the results of decreasedsplenomegaly, treatment of FV-infected mice with Δ¹²-PGJ₃ led to thebetter demarcation of peri-arteriolar lymphoid tissue (FIG. 2D). Theerythroid progenitor cells were substantially lower and a few giantcells were seen accompanied by an increase in the number of apoptoticbodies with increased individual tumor cell necrosis in the CyPG treatedgroup when compared to the vehicle-treated FV-infected group (FIG. 2D).CyPG treatment of FV-infected mice restored the splenic architecture,with well-defined red and white pulp regions, as in the uninfected mice.

Δ¹²-PGJ₃ inhibits the expansion of LSCs, but not the viral replication.To rule out the possibility that Δ¹²-PGJ₃ blocks FV-induced leukemia byinhibiting viral replication, a second model of FV-induced leukemia wasused. Here, the FV-LSCs were transplanted into Stk^(−/−) mice.Short-form Stk (Sf-Stk), a naturally occurring truncated form of Stk/Ronreceptor tyrosine kinase, is encoded by the FV-susceptibility locus 2(Fv2) (Persons DA et al., Nat Genet. 1999;23:159-165). Fv2 resistantmice express low levels of Sf-Stk, which fails to support theproliferation of infected cells. Thus, transplantation of FV-LSC intoStk^(−/−) mice results in leukemia caused by the expansion of donorcells and not by the spread of viral infection. LSCs generated from wildtype mice were transplanted into syngeneic Stk^(−/−) mice. Treatmentwith Δ¹²-PGJ₃ (at 0.025 mg/kg and 0.05 mg/kg) led to significantlydecreased splenomegaly with a concomitant decrease in leukocyte counts(FIGS. 3A-C). Flow cytometric analysis of LSCs in the spleens oftransplanted Stk^(−/−) mice indicated complete ablation of M34⁺Sca1⁺Kit⁺cells upon treatment with Δ¹²-PGJ₃ (FIG. 3D); while the LSCs fromStk^(−/−) mice treated with 9,10-dihydro-15d-PGJ₂ or the vehicle did nothave any effect on their viability nor alleviated splenomegaly.Treatment of FV-induced leukemia with Δ¹²-PGJ₃ or 15d-PGD₂ significantlydecreased the hematocrit, WBC counts, and reticulocyte counts that areall hallmarks of leukemia; while 9,10-dihydro-15d-PGJ₂ had no effect onany parameter tested above (FIG. 3C).

Δ¹²-PGJ₃ alleviates leukemia caused by transplantation of BCR-ABL⁺LSCs.The in vitro studies of FIG. 1 showed that Δ¹²-PGJ₃ treatment causedapoptosis of BCR-ABL⁺LSCs, but not the normal HSCs (MSCV-GFP⁺HSCs).Next, the anti-leukemic activity of Δ¹²-PGJ₃ in BCR-ABL mice, which is amodel for the chronic phase of CML (Pear WS et al., Blood.1998;92:3780-3792), was examined. As shown in FIG. 4, treatment of micetransplanted with BCR-ABL⁺LSC with 0.05 mg/kg of Δ¹²-PGJ₃ for 1 weekcompletely alleviated splenomegaly with spleen weights close to thosetransplanted with the MSCV-GFP⁺ HSCs (FIG. 4A). Furthermore, Δ¹²-PGJ₃treatment also significantly decreased the leukocyte counts in theperipheral blood (FIG. 4B), decreased Kit⁺Sca1⁺GFP⁺LSCs in the spleen(FIG. 4C), as well as eradicated Kit⁺ Sca1⁺ Lin⁻ GFP⁺ LSCs in the bonemarrow of the BCR-ABL⁺LSC transplanted mice (FIG. 4D). More importantly,treatment of BCR-ABL⁺LSC transplanted mice with Δ¹²-PGJ₃ rescued all ofthe mice; while those treated with vehicle died two weeks aftertransplantation of LSCs (FIG. 4E). In contrast, treatment of micetransplanted with MSCV-GFP⁺ HSC with Δ¹²-PGJ₃ had no effect on WBCcounts or other hematological parameters or survival, suggesting thatΔ¹²-PGJ₃ does not affect steady state hematopoiesis (FIG. 4E). Tofurther demonstrate that Δ¹²-PGJ₃ does not affect normal hematopoieticdifferentiation, it was next tested whether Δ¹²-PGJ₃ treatment had anadverse effect on hematopoietic progenitors by testing its effect oncolony forming ability in CFC assays. Bone marrow from 5-FU treated micewas plated in methylcellulose media containing multiple cytokines in theabsence or presence of 25 nM of Δ¹²-PGJ₃. There was no difference in thenumber of CFC in Δ¹²-PGJ₃ treated compared to control (PBS)-treatedcells (FIG. 4F).

In order to confirm that Δ¹²-PGJ₃ had eradicated LSCs, secondarytransplants using splenocytes from Δ¹²-PGJ₃ or vehicle treated BCR-ABLmice were performed. The original donor MIGR-BCR-ABL transduced bonemarrow cells were marked with CD45.1⁺; while the primary and secondaryrecipients were CD45.2⁺. Secondary transplants that received donor cellsfrom vehicle-treated mice rapidly developed splenomegaly and high WBCcounts indicative of leukemia. In contrast, second recipients of donorcells from Δ¹²-PGJ₃-treated mice failed to develop splenomegaly or highWBC counts (FIG. 5A). Further analysis of spleen for LSCs showed thatrecipients of donor cells from Δ¹²-PGJ₃-treated mice lackedKit⁺Sca1⁺GFP⁺ cells. In addition, analysis of CD45.1 expression alsoshowed that CD45.1⁺ donor cells were not present in the spleen (FIG.5C). Secondary recipients of donor cells from vehicle-treated miceexhibited large numbers of donor-derived Kit⁺Sca1⁺GFP⁺ and CD45.1⁺ donorcells in their spleens (FIG. 5B and C). Similar secondary transplantexperiments were performed with donor spleen cells isolated from FV-LSCtransplanted BALB/c-Stk^(−/−) mice treated with Δ¹²-PGJ₃ or vehicle.Similar to the BCR-ABL secondary transplants, mice receiving donor cellsfrom Δ¹²-PGJ₃ treated mice failed to develop splenomegaly or high WBCcounts and lacked LSCs in their spleens (FIG. 5D and E). Taken together,these data clearly demonstrate the ability of Δ¹²-PGJ₃ to eradicate LSCsin two diverse murine models of myeloid leukemia.

EPA-metabolites selectively activate p53 in LSC: Ex-vivo treatment ofsorted LSCs from FV-infected mice with 10 or 25 nM of Δ¹²-PGJ₃ for 12 hled to significant upregulation of p53 expression at the transcriptlevel. Similarly, treatment of LSCs with 15d-PGJ₂ also showed a similartrend; while 9,10-dihydro-15d-PGJ₂ was ineffective. Interestingly, PGJ₂treatment upregulated the expression of p53 to only a minor degree, butnot to the extent observed with other CyPGs. However, preincubation ofPGJ₂ with media (at 37° C.) for 42 h prior to addition of LSCs led toincreased expression of p53, which suggests a time-dependentisomerization event that possibly converts PGJ₂ (Δ¹³-PGJ₂) to Δ¹²PGJ₂ or15d-PGJ₂ and PGJ₃ (Δ¹³-PGJ₃) to Δ¹²-PGJ₃ or 15d-PGJ₃ that makes thelatter metabolites more potent than the precursor (FIG. 1E). Treatmentof BCR-ABL⁺ LSCs ex vivo with Δ¹²-PGJ₃ or 15d-PGJ₂ (25 nM) also led to asimilar increase in p53 mRNA; while 9,10-dihydro-15d-PGJ₂-treatment wasineffective. Time course analysis showed that p53 transcript levelsrapidly increased following treatment of FV-LSCs ex vivo with Δ¹²-PGJ₃such that by 12 h maximal p53 expression was observed. Analysis of p53expression in total spleen of uninfected and vehicle-treated FV infectedmice clearly showed no increase; however Δ¹²-PGJ₃-treated mice (treatedfor 1 week) showed a significant expression of p53 in the spleen.Similarly, an increase in the nuclear levels of p53 protein was observedin sorted LSC treated with Δ¹²-PGJ₃ for 12 h, but not in the untreatedor vehicle-treated cells. To confirm the role of p53 as a criticalmediator of Δ¹²-PGJ₃-dependent LSC apoptosis, the pro-apoptotic role ofCyPGs was examined in murine erythroleukemia (MEL) cells that lackfunctional p53. MEL cells are derived from CFU-FV that has been expandedinto a cell line. Treatment of MEL cells with Δ¹²-PGJ₃ failed toinitiate apoptosis. MEL cells exhibited sensitivity to treatment withanti-leukemic drugs such as daunorubicin, mitoxantrone, and cytarabine,but not to nutlin, a small molecule inhibitor of MDM2-p53 interactionthat causes reactivation of p53. These data confirm the role ofΔ¹²-PGJ₃-dependent activation of the p53 pathway in apoptosis of LSCs.

The activation of p53 activity is known to be regulated by anATM-dependent signaling pathway. It was next examined if ATM played acritical role in the pro-apoptotic activity of Δ¹²-PGJ₃. In addition toincreased phosphorylated-p53 protein, an increase in the levels of pChk2was observed only in the total spleen extracts from the Δ¹²-PGJ₃-treatedmice transplanted with FV-LSCs. TUNEL staining of splenic sections fromFV-infected mice showed increased apoptosis only in the Δ¹²-PGJ₃-treatedgroup. In agreement with the TUNEL staining results, activation of Baxexpression was observed, which is a downstream mediator of apoptosis inthe spleens of Δ¹²-PGJ₃-treated FV-infected mice, but not theFV-infected vehicle-treated mice. Taken together, the above experimentssuggested the involvement of ATM-p53 signaling axis in promotingΔ¹²-PGJ₃-dependent apoptosis. To further confirm the involvement of ATM,two well characterized inhibitors of ATM were utilized. Preincubation ofsorted FV-LSC ex vivo with a high-affinity inhibitor of ATM (MTPO) aswell as a dual inhibitor of ATM and the related ATR kinase (CGK-733)followed by treatment with Δ¹²-PGJ₃ blocked the CyPG-dependentexpression of p53, which indicated that ATM served as a criticalmediator of apoptosis by Δ¹²-PGJ₃. Similar to what was observed with FV,treatment of BCR-ABL⁺ LSCs (Kit⁺Sca1⁺Lin⁻ GFP⁺) with 25 nM of Δ¹²-PGJ₃led to a significant increase in apoptosis as seen by increased annexinV staining and western blot analysis of caspase 3 and caspase 8activation. Δ¹²-PGJ₃ treatment led to an increase in p53 transcription;while there was a concomitant decrease in GFP⁺ cells. Similar to whatwas observed in FV-LSCs, pretreatment of these cells with MTPO blockedthe effect of Δ¹²-PGJ₃ on apoptosis and induction of p53 expression.

DISCUSSION

In the present study, the metabolism of EPA-derived PGD₃ tocyclopentenone PGJ₃, Δ¹²-PGJ₃, and 15d-PGJ₃ in macrophages wasdemonstrated. Of these stable metabolites that were detected in themacrophage culture media, only Δ¹²-PGJ₃, and 15d-PGJ₃ targeted LSCs forapoptosis in FV-induced leukemia and BCR-ABL⁺ retrovirus-based murinemodel of CML. In contrast, EPA and PGJ₃ were ineffective. These datasuggest a structure-function relationship in the form of analkylidenecyclopentenone structure with an unsaturation at carbon-12 asa requirement for the apoptotic activity of CyPGs.

Based on LC-MS/MS studies, a sufficient quantity of Δ¹²-PGJ₃ (˜0.15μM/10⁶ macrophages) was produced by macrophages that were well withinthe concentration required to cause apoptosis of LSCs (IC₅₀=7 nM).Despite its pro-apoptotic effects on LSCs, Δ¹²-PGJ₃ had no adverseeffects on HSCs or downstream progenitors. These studies indicate thatLSCs exhibit increased sensitivity to Δ¹²-PGJ₃ and other related CyPGsin a stereoselective manner. The induction of apoptosis in LSCs by theseendogenous metabolites requires the ATM/p53 signaling axis, which causescomplete ablation of leukemia in-vivo, as seen in two different mousemodels of leukemia. These data show that treatment eliminates LSCs tosuch an extent that no LSC activity is observed on secondary transplant.These studies support the role of ATM as an important mediator ofelectrophilic “stress” response pathway in LSCs.

In summary, the ability of macrophages to produce endogenous Δ¹²-PGJ₃that displays potent proapoptotic activity towards LSCs was demonstratedin two murine models of leukemia by activating the ATM-p53 pathway ofapoptosis. Intraperitoneal administration of Δ¹²-PGJ₃ eradicated LSCs ina BCR-ABL retroviral model of CML with no relapse noted five weeks postadministration of last dose of Δ¹²-PGJ₃. In contrast, vehicle-treatedmice transplanted with LSC failed to survive past day 16post-transplantation (FIG. 4F). Current anticancer therapies areineffective against LSCs; thus the ability of a stable endogenousmetabolite to ablate LSCs identifies it as a potential therapy. Inaddition, these results indicate that Δ¹²-PGJ₃, derived from dietary n-3PUFA, has the potential to serve as a chemopreventive agent in thetreatment of leukemia.

Supplementary Information and Methods

Preparation, isolation, and spectroscopic characterization of PGD₃metabolites. PGD₃ (Cayman Chemicals) was incubated with 0.1 M sodiumphosphate buffer, pH 7.4, containing 0.9% NaCl at a final concentrationof 100 μg/ml with shaking at 37° C. for varying periods (24 h-144 h) insealed brown vials flushed with argon. Similar reactions were performedin the presence of 10% FBS. The reaction mixtures or cell culture mediasupernatants were acidified with 1 N HCl to pH 3.0 and extracted threetimes with two volumes of hexane:ethylacetate (50:50). The organic phasewas passed over anhydrous sodium sulfate and evaporated under argon. Theorganic phase was stored in −80° C. until further processing.Eicosanoids were separated by reverse phase LC on a Dynamaxsemi-quantitative C₁₈ column (10×250 mm) using MeCN: H₂O: acetic acid(70:30:1) at 2 ml/min and the eluate was monitored at 280 nm. The peakswere collected, concentrated using argon and reconstituted in MS-grademethanol for MS/MS and UV spectroscopic analysis. Eicosanoids wereanalyzed by direct infusion into a triple quadruple mass spectrometer(API 2000, ABI SCIEX) in the negative electrospray ionization mode. Theelectrospray voltage and ion spray source temperature were set to −4000V and 300° C., respectively. Nitrogen was used as curtain (12 psi) andnebulizer (15 psi) gas. The declustering, defocusing, and entrancepotentials were set at −50 V, −400 V, and −10 V, respectively.

Δ¹²-PGJ₃ purified by HPLC was used to create a standard calibrationcurve on the MS operated in multiple reaction-monitoring (MRM) mode fortwo transitions, 331.5 to 313.5 m/z and 331.5 to 269.5 m/z. UV spectraof all LC-purified PGD₃ metabolites in methanol were recorded on aBeckman DU7500 Diode Array Spectrophotometer. The molar extinctioncoefficients for PGJ₂, Δ¹²-PGJ₂, and 15d-PGJ₂ (all from CaymanChemicals) were used to calculate the concentrations of PGJ₃, Δ¹²-PGJ₃,and 15d-PGJ₃, respectively.

Semiquantitative RT-PCR for p53 and β-actin. Semiquantitative-PCR wasperformed on the cDNA prepared from LSCs. The bands were visualized onan agarose (1% w/v) gel and evaluated by densitometry.

Apoptosis. The LSCs were diluted in DMEM, resuspended using a 16-gaugeneedle, and collected by centrifugation. 1×10⁵ cells were resuspended in200 μl of binding buffer (0.1 M HEPES with 1.4 M NaCl, 25 mM CaCl₂, pH7.4). Annexin V FITC (BD Biosciences) was incubated with cells for 15min on ice followed by flow cytometric analysis.

Cell viability studies. MEL cells were cultured in DMEM containing 10%FBS and treated with various commonly used anti-leukemic drugs such asdaunorubicin (DNR), mitoxantrone (MIT), and cytarabine (CYT) at a finalconcentration 1 μM for 24 h. Nutlin (5 μM; Cayman Chemicals), a p53activator, was used as a control to demonstrate the lack of activationof p53 and apoptosis in the MEL cells. After 24 h of drug treatment,cell proliferation was measured by MTT assay with CCK-8 kit from DojindoMolecular Technologies, Inc. (Gaithersburg, Md.). All viability valuesreported are relative to untreated cells (UT) that was designated to be100%. Results represent the mean±SEM of three independent observations.

Referring to FIG. 6, spontaneous conversion of PGD₃ to PGJ₃, Δ¹²-PGJ₃,and 15d-PGJ₃ in-vitro is shown. FIG. 6 a is a schematic showing thepathway of conversion of EPA to CyPGs. Representative MS of PGD₃ and15d-PGJ₃ are shown. In FIGS. 6 b-d, PGD₃ (from Cayman Chemicals) wasincubated with 0.1 M sodium phosphate buffer, pH 7.4, containing 0.9%NaCl at a final concentration of 100 μg/ml with shaking at 37° C. forvarying periods (24 h-144 h) in sealed brown vials flushed with argon.In FIG. 6 e, PGD₃ was incubated as above in 10% FBS diluted in phosphatebuffered saline for 48 h at 37° C. The PGs were extracted usinghexane:ethylacetate (50:50) and the organic phase was concentrated withargon. The eicosanoids were separated by reverse phase LC on a Dynamaxsemi-quantitative C₁₈ column (10×250 mm) using MeCN: H₂O: acetic acid(70:30:1) at 2 ml/min and the eluate was monitored at 280 nm. The peakswere collected, concentrated using argon and reconstituted in MS-grademethanol for UV-MS/MS analyses. Representative of N=8 independentreactions.

A UV-Spectroscopic analysis of Δ¹²-PGJ₃ and 15d-PGJ₃ as a function oftime during conversion was performed. LC-purified Δ¹²-PGJ₃ and 15d-PGJ₃were reconstituted in methanol and analyzed by UV spectroscopy forspectral properties on a Beckman DU7500 Diode Array Spectrophotometeragainst appropriate solvent background controls. The molar extinctioncoefficients for PGJ₂, Δ¹²-PGJ₂, and 15d-PGJ₂ were used to calculate theconcentrations of PGJ₃, Δ¹²-PGJ₃, and 15d-PGJ₃, respectively.Representative of N=3. The results indicate the formation of analkylidenecyclopentenone structure followed by a intramolecularrearrangement to form Δ¹²-PGJ₃ and the double dehydration product15d-PGJ₃ from PGD₃ precursor.

Referring to FIG. 7, a dose-dependent pro-apoptotic effect of CyPGs onLSCs is shown. The ability of CyPGs derived from arachidonic acid (2series PGs) to induce apoptosis was tested in cultures of LSCs from FVinfected mice and in the murine CML model. In FIG. 7A, Δ¹²-PGJ₂, or15d-PGJ₂ are able to induce apoptosis, compared to vehicle, but PGJ₂ wasnot. 15d-PGJ₂ is known as a PPAPy agonist. In FIG. 7B, FV LSCs aretreated with 15d-PGJ₂ , an inactive form, 9,10-dihydro-15d-PGJ₂ orRosiglizone, a commercially available synthetic PPAPγ agonist (inset ofFIG. 7B). Rosiglizone and 9,10-dihydro-15d-PGJ₂ fail to induceapoptosis. In FIG. 7C Δ¹²-PGJ₃, 15d-PGJ₂ or 9,10-dihydro-15d-PGJ₂ aretested for their ability to induce apoptosis in a murine CML model. OnlyΔ¹²-PGJ₂, 15d-PGJ₂ are active, while 9,10-dihydro-15d-PGJ₂ had noeffect. These data show that activation of PPAPy is not the mechanism bywhich CyPGs induce apoptosis. In FIG. 7 a, Spleen cells from FV-infectedmice were sorted for M3⁴⁺Sca¹⁺Ki^(t+) LSCs and incubated with 25 nM ofPGJ₂, Δ¹²-PGJ₂, or 15d-PGJ₂ for 36 h in a methylcellulose stem cellmedia with 200 ng/ml sonic hedgehog (sHH), 50 ng/ml SCF, and 15 ng/mlBMP4. The cells were stained with annexin V-FITC and analyzed by flowcytometry. Representative of N=4. Means±s.e.m. *P<0.001 compared tovehicle (PBS). In FIG. 7 b, a comparison of the proapoptotic function of9,10-dihydro-15d-PGJ₂ with 15d-PGJ₂ is shown. Inset: Effect ofrosiglitazone on the apoptosis of LSC. Rosiglitazone (0.1-2.0 μM) wasincubated with LSC in the culture media for 36 h as described earlierand the cells were subjected to annexin-V staining followed by flowcytometry. FIG. 7 c shows results from an analysis of apoptosis ofBCR-AB^(L+)Ki^(t+)Sca¹⁺ cells isolated from the spleens of micetransplanted with BCR-AB^(L+) transduced HSCs after treatment withCyPGs. LSC were treated ex vivo with indicated concentrations of eachcompound for 36 h. Mean±s.e.m. shown *P<0.001.

The effect of Δ¹²-PGJ₃ on NF-κB and PPAPγ was examined. RAW264.7macrophages were pretreated with DMSO, Δ¹²-PGJ₃ at 0.25, or 1.0 μM andsubsequently stimulated with 100 ng/mL E.coli LPS for 4 h. The nuclearextracts were prepared and the binding of NΦκB to a ³²P-labeledconsensus double stranded oligonucleotide probe was examined using gelshift analysis. NS=non-specific band. Lanes 1-5 represent untreated, LPSalone, DMSO+LPS, Δ¹²⁻PGJ₃ (0.25 μM)+LPS, and Δ¹²-PGJ₃ (1 μM)+LPS,respectively. BCR-ABL LSCs were sorted from spleens, plated, and treatedwith PBS, 25 nM Δ¹²-PGJ₃, or 9,10-dihydro 15d-PGD₂ for 0, 2 or 6 h. Thecells were harvested and nuclear extracts were prepared using standardtechniques. Ten μg of nuclear protein was used from each sample for thegel-shift reaction. As a positive control for NF-κB, nuclear extractfrom LPS-treated murine (RAW264.7) macrophages was used. Anti-p50 Ab wasused with this positive control for a supershift, and excess ‘cold’probe was used with the positive control as a ‘cold competitor’. AWestern blot analysis was performed of the above-mentioned nuclearextracts from BCR-ABL LSCs treated with PBS, Δ¹²-PG₃₃ or9,10-dihydro-15d-PGJ₂ for various time periods (0-6 h) probed withanti-p65 and anti-β-actin antibodies. In a reporter assay for PPAPγactivation, HEK293T cells expressing ligand-binding domain of humanPPAPy fused to yeast GAL4 DNA binding domain were transfected with apGa1RE-Luc reporter gene. These studies were performed to address theability of Δ¹²-PGJ₃ to activate PPAPγ using this well characterizedreporter system. Our studies demonstrated that Δ¹²-PGJ₃ was unable toactivate the PPAPy at concentrations 0.01 μM to 1.0 μM, unlikerosiglitazole that was used as a positive control.

Treatment of mice transplanted with FV LSCs with Δ¹²-PGJ₃ (0.05 mg/kg)does not adversely affect hematological parameters in the mice. Completeblood counts of mice treated with Δ¹²-PGJ₃ (0.05 mg/kg) were examined.There is no difference in treated mice and untransplanted control micein terms of white blood cell counts, red blood cell counts or plateletcounts. Changes in hematological parameters in FV-infected mice upontreatment with CyPGs were examined. FV-infected Balb/c mice were treatedwith Δ¹²-PGJ₃ (0.05 mg/kg) intraperitoneally for 7 d following which themice were sacrificed and hematological parameters were analyzed on anAdvia blood analyzer. FV-infected Δ¹²-PGJ₃ treated mice were compared toinfected vehicle controls. N=5 per group, all data are means±s.e.m.*P<0.05. 3% w/v hydroxypropyl-β-cyclodextrin was used as a vehicle in invivo experiments. Spleen sizes of FV-infected mice that were treatedwith either vehicle, 9,10-dihydro-15d-PGJ₂ (0.05 mg/kg), or 15d-PGJ₂(0.05 mg/kg) were examined; N=3 pre group. Hematological parameters ofBalb/c uninfected, infected, and 15d-PGJ₂ treated mice were examined.N=5 per group. All data were means±s.e.m. *P<0.05.

15d-PGJ₂ was shown to eradicate FV-LSC. FV-LSC were targeted by 15d-PGJ₂in the spleen of FV-infected mice. FV-infected mice were treated with15d-PGJ₂ or 9,10-dihydro-15d-PGJ₂ at 0.05 mg/kg for 7 d. The splenic LSC(M34⁺Sca1⁺Kit⁺) cells were analyzed by flow cytometry on day 14 postinfection. Treatment with 15d-PGJ₂ leads to a significant decrease inLSC numbers as measured by flow cytometry. In addition, 15dPGJ₂significantly decreased the number of transformed leukemia cells thatare capable of forming transformed CFU-Friend virus colonies. 15d-PGJ₂does not affect Friend virus viral replication, so in these experimentsa single course of treatment with 15dPGJ₂ does not eliminate LSCs, whichare regenerated by ongoing viral infection. Uninfected and infectedvehicle controls were used for comparison. N=3; *P<0.05. Splenocytesfrom infected mice were treated with vehicle, 9,10-dihydro-15d-PGJ₂, and15d-PGJ₂, and were plated in methylcellulose media containing FCSwithout growth factors to examine if treatment of mice with 15d-PGJ₂ or9,10-dihydro-15d-PGJ₂ affected the formation of CFU-FV colonies, whichexhibit factor-independent growth. The colonies were counted 10-14 daysafter plating. N=3 mice per group, *p<0.05.

In order to address the ability of 15d-PGD₂ to eradicate FV LSCs in asystem where viral infection cannot regenerate LSCs Stk^(−/−) mice weretransplanted with in vitro expanded FV-LSCs. Stk^(−/−) mice areresistant to Friend virus infection so the leukemia developed by thetransplanted mice is result of the transplanted LSCs and not FVinfection. Mice treated with 15dPGJ₂ led to the eradication of FV LSCsin the spleen and resolution of the diseases. LSCs sorted from thespleens of FV-infected mice were transplanted into Stk^(−/−) mice (on aBalb/c background). After 6 weeks such mice were treated daily for 1week with vehicle (hydroxypropyl-β-cyclodextrin), 15d-PGJ₂ (0.05 mg/kg),or 9,10-dihydro-15d-PGJ₂ (0.05 mg/kg) by intraperitoneal injection. Themice were sacrificed 51 days post LSC transplantation for analysis. Ananalysis of spleens of mice comparing splenomegaly in vehicle, 15d-PGJ₂,or 9,10-dihydro-15d-PGJ₂ treated transplanted mice was performed. Spleenweight compared to control (untransplanted mice) after treatment and WBCcounts in the peripheral blood of the mice after treatment wereexamined, and a flow cytometric analysis of the spleen of untransplantedand LSC transplanted mice after treatment was performed. All data weremean±s.e.m. *p<0.05 compared to control or 9,10-dihydro-15d-PGJ₂ treatedgroups. N=5 per group.

Δ¹²-PGJ₃ cannot induce apoptosis in MEL cells because MEL cells have amutation in the p53 gene. In order to address whether MEL cells areresistant in general to chemotherapy agents, we tested whether MEL cellscan be killed by apoptosis when treated with standard anti-leukemiadrugs. Treatment with with various commonly used anti-leukemic drugssuch as daunorubicin (DNR), mitoxantrone (MIT), and cytarabine (CYT) ata final concentration 1 μM for 24 h. Nutlin (5 μM), a p53 activator, wasused as a control to demonstrate the lack of activation of p53 andapoptosis in the MEL cells. After 24 h of drug treatment, cellproliferation was measured by MTT assay with CCK-8 kit from DojindoMolecular Technologies, Inc. (Gaithersburg, Md.). All compounds with theexception of nutlin caused significant apoptosis. The results representthe mean±SEM of three independent observations.

Example 2 Targeting LSCs Via Molecules That Activate DP

The data shown in FIGS. 8 and 9 suggests a role of a class of G-proteincoupled receptors (called DP), which play a role in the apoptosis ofLSCs by Δ¹²-PGJ₃. In addition, experiments were performed with syntheticagonists of the receptor as well.

Referring to FIG. 8, this data shows that imatinib-resistantBCR-ABL(GFP)+ cells Δ¹²-PGJ₃. are targeted by A In this experiment, micewere transplanted with BCR-ABL+ LSCs and a week later imatinib treatmentwas initiated by i.p. for 1 wk at 25 mg/kg/day. After a 1 week washoutperiod post imatinib treatment, spleens were dissected and the totalsplenocytes were treated ex-vivo with 25 nM Δ¹²-PGJ₃ or vehicle (PBS)for 24 h. GFP+ LSCs were analyzed by flow cytometry. As shown in FIG. 8,Δ¹²-PGJ₃ treatment can even target LSCs that are resistant to imatinibtreatment.

An experiment was performed that showed apoptosis of BCR-ABL+ LSCs byΔ¹²-PGJ₃ is inhibited by synthetic antagonists of the DP. In thisexperiment, sorted BCR-ABL+LSCs and MSCV-HSCs cultured in methocultmedia were pretreated with MK0824 (DP1 antagonist; 10 nM; Cayman Chem),CAY10471 (DP2 antagonist; 10 nM; Cayman Chem), or KU55933 (ATM kinaseinhibitor; 10 nM; Calbiochem) for 2 h followed by 25 nM Δ¹²-PGJ₃ orDMSO. Following 36 h of incubation, apoptotic cells were quantified byannexin V staining. Viability of the MSCV-HSC control cells were notaffected by any of the above treatments. Mean±s.e.m. of n=3. CAY10471 isan analog of Ramatroban (a approved human medication for the treatmentof allergic rhinitis), which contains modifications that increase bothits potency and selectivity for the human CRTH2/DP2 receptor. CAY 10471binds to the human CRTH2/DP2, DP1, and TP receptors with Ki values of0.6, 1200, and >10,000 nM, respectively. MSCV-HSCs (normal HSCs) werenot affected by any of the treatments above. BCR-ABL LSCs on the otherhand are highly susceptible to apoptosis by 25 nM Δ¹²-PGJ₃ and such aneffect is inhibited by the use of DP antagonists. From this data, thepathway of apoptosis involves activation of DP and ATM kinase (Ataxiatelangiesctasia mutated kinase protein). In a related study to addressif Δ¹²-PGJ₃ treatment would cause any degranulation of granulocytes, arat basophilic cell line (RBL-23) was treated with 100 nM Δ¹²-PGJ₃ andthe degranulation was followed by quantitating the release of histamineand a second marker of degranulation, hexoseaminidase. Our resultsclearly indicate that Δ¹²-PGJ₃ did not cause degranulation, whileionomycin, a well-known stimulant of degranulation, caused extensiveproduction of histamine and hexoseaminidase.

Referring to FIG. 9, this data shows apoptosis of BCR-ABL+ LSCs bysynthetic agonists of the DP. In this experiment, 500,000 BCR-ABL+ LSCswere plated in a 24-well plate followed and were treated with 25 nMPGD₂Me or 100 nM ZK118182 (both are agonists of DP) for 24 h. GFP+ cellswere analyzed by flow cytometry. These agonists were purchased fromCayman Chemicals, Mich. Based on this and the previous data, it is veryclear that DP activation by synthetic agonists can induce apoptosis ofLSCs. Thus, the use of synthetic compounds that are well-established DPagonists can target LSCs.

Example 3 Δ¹²-PGJ₃ and Related Agonists Do Not Affect Normal HumanHematopoiesis

Referring to FIG. 10, this graph shows that Δ¹²-PGJ₃ and relatedagonists do not affect normal human hematopoiesis. In an experimentdescribed above, the data showed the effects of Δ¹²-PGJ₃ on theformation of terminally differentiated hematopoietic cell colonies.These colonies are called colony forming cells or CFC. For thatexperiment, only growth factors necessary for multilineage myeloidcolony formation were added. Referring to FIG. 10, media containingmultiple growth factors supplemented with the compounds listed on the Xaxis of the graph was used. Δ¹²-PGJ₃ had no effect. The synthetic DPagonist ZK also had no effect. In conclusion, Δ¹²-PGJ₃ and relatedagonists do not affect normal human hematopoiesis.

Referring to FIG. 11, Δ¹²-PGJ₃ does not affect the ability of normalbone marrow cells to differentiate (to form BFUe). In this experiment,human bone marrow cells (CD34+; Reach Bio, Seattle, Wash.) were culturedin Methylcellulose (Stem Cell Technology, H-4230) with Epo (3 U/ml)+SCF(50 ng/ml) for 8 days with PBS, Δ¹²-PGJ₃ (50 nM and 100 nM). BFUecolonies were stained with benzidine stain on day 8.

Example 4 Efficacy of Δ¹²-PGJ₃ and Comparative Data to Imatinib

An experiment involving a cytospin of Blast crisis CML (011711) andGeimsa stain was performed. Blast crisis CML were cultured in IMDM withBIT, LDL, L-Glu and treated with PBS or 100 nM Δ¹²-PGJ₃ for 12 hrs.Cells were collected and slides were done by Cytospin. Cytospin slideswere stained with Wright Geimsa stain and pictures (100×) were recordedon a Material microscope. The studies confirm death of blast-crisis CMLcells.

Referring to FIG. 12, the data in this pair of graphs shows that DPmediate the Δ¹²-PGJ₃-dependent apoptosis of blast crisis CML cells froma patient (#011711). In this experiment, 110,000/well primary AML cellswere cultured in above specified media for 6 and 12 hrs. Cells werecollected and washed once with ice cold PBS. The cells were resuspendedin 200 ul 1× Apoptosis buffer with annexin-V PE to all tubes. The cellswere washed in PBS and resuspended in 600 ul PBS and transfer into flowtubes and analyzed for apoptosis (Annexin V+ cells) on FC-500. Theconclusion of this experiment is that Δ¹²-PGJ₃ or a synthetic DP agonistinduces apoptosis in BC-CML primary patient cells. DP agonists blockthis response demonstrating that the effect of Δ¹²-PGJ₃ is DP dependent.Referring to FIG. 13, this figure shows that DP mediate theΔ¹²-PGJ₃-dependent apoptosis of AML cells from a patient (#100810). Inthis experiment, 110,000/well AML cells were cultured in earliermentioned media for 6 and 12 hrs. Cells were collected and washed oncein PBS and resuspended in 200 ul PBS and blocked with FC receptorantibody 10 mins RT. The following antibodies were added: CD38, CD123,CD34, CD117 (BD bioscience) for 1 hr on ice. Cells were washed in PBSonce and resuspended in 200 ul of apoptotic buffer, and annexin V wasadded and incubated for 15 mins. Apoptotic cells (Annexin V+ cells) werecounted by flow cytometry. The conclusion from this experiment is thatΔ¹²-PGJ₃ induces apoptosis of primary human AML cells and that it canspecifically kill LSCs as measured by analyzing the Annexin V+ fractionof the CD34+CD38−CD123+CD117+cells. Similar studies were performed withother AML patient samples (AML patient #123009, #033107, #041909,#101308) and the results (see FIG. 15) were identical to that describedabove. Δ¹²-PGJ₃ targeted the LSCs in all these samples. Moreimportantly, pre-treatment of LSCs with CAY10471 (DP antagonist)completely blocked the apoptosis by Δ¹²-PGJ₃. Referring to theexperimental results shown in FIG. 15, 110,000/well AML cells frompatients #100810, 123009, 033107, 041909, 101308 were cultured inearlier mentioned media for 6 h with or without 100 nM Δ¹²-PGJ₃ (100nM), or pretreatment with CAY10471 (10 nM) followed by Δ¹²-PGJ₃ (100nM). Cells were collected and washed once in PBS and resuspended in 200ul PBS and blocked with FC receptor antibody 10 mins RT. The followingantibodies were added: CD38, CD123, CD34 (BD bioscience) for 1 hr onice. Cells were washed in PBS once and resuspended in 200 ul ofapoptotic buffer, and annexin V was added and incubated for 15 mins.Apoptotic cells (Annexin V+ cells) were counted by flow cytometry. Theconclusion from this experiment is that Δ¹²-PGJ₃ induces apoptosis ofprimary human AML cells and that it can specifically kill LSCs asmeasured by analyzing the Annexin V+ fraction of theCD34+CD38−CD123+cells.

Survivin expression in the human AML sample post Δ¹²-PGJ₃ treatments wasexamined. Total RNA was isolated from AML cells with indicatedtreatments (for 6 h) using Trizol (Invitrogen) cDNA was generated usingSuperscript II (Invitrogen) and cDNA were quantified by RT-PCR usingSYBR green PCR master mix and primers that amplify a 76 nt PCR product.A Taqman probe for GAPDH (Applied biosystems) was used. Survivin is aninhibitor apoptosis. Δ¹²-PGJ₃ decreases the expression of survivinsuggesting that DP agonists suppress counter-regulatory pathways thatinhibit apoptosis.

MCL-1 expression in Δ¹²-PGJ₃ treated AML was examined. MCL-1 is anantiapoptotic gene that belongs to the Bc1-2 family. Total RNA and cDNAwere isolated from primary AML cells as indicated above. MC1-1expression was measured using real time PCR. (Hs01050896-ml, AppliedBiosystems). Treatment with Δ¹²-PGJ₃ decreases the expression MCL1 whichis associated with increased apoptosis.

Referring to FIG. 14, this graph shows a comparison of Δ¹²-PGJ₃ withImatinib (Gleevec) in the BCR-AB^(1/+)LSC transplant CML model in mice.In this experiment, Imatinib and Δ¹²-PGJ₃ were used at 75 mg/kg and0.025 mg/kg, respectively. Treatment of a murine model for CML with thestandard of care for CML patients, which is Imatinib therapy for 1 weekleads to prolonged survival, but rapid relapse of leukemia. In contrasttreatment with Δ¹²-PGJ₃ leads to prolonged survival, but no relapse ofleukemia.

Example 5 Δ¹²-PGJ₃ as an AML Chemotherapeutic

AML is one of the most common types of leukemia in adults.Unfortunately, the five year relative survival rates for AML are thelowest when compared to other forms of leukemia. AML is a stem celldisease where LSCs occupy the apex of the disease hierarchy. LSCs canself renew and generate non-stem cell progeny that make up the bulk ofthe leukemia cells. Although chemotherapy agents can effectively targetbulk leukemia cells, LSCs have active mechanisms to avoid killing bythese drugs. As a consequence, failure to eliminate LSCs results inrelapse of the disease. Because of this property, specific targeting ofLSCs is essential for successful treatment. Although the need for newanti-LSC based therapies is well recognized, the identification ofmechanism-based drugs to target LSCs has been lacking. Clearly newapproaches are needed. A metabolite derived from ω-3 fatty acids,Δ¹²-PGJ₃, was discovered which effectively eradicates LSCs in two mousemodels of chronic leukemia. In the experiments described herein, thesefindings were extended to show that Δ¹²-PGJ₃ effectively targets AMLLSCs by inducing apoptosis in murine models of AML and in human AMLleukemia samples. In contrast, Δ¹²-PGJ₃ has no effect on normalhematopoietic stem cells or the differentiation of hematopoieticprogenitors. Δ¹²-PGJ₃ acts by inducing the expression of p53 in LSCs andleukemia cells. High-level expression of p53 in LSCs is incompatiblewith self renewal and leads to apoptosis. These data suggest thatΔ¹²-PGJ₃ is a chemotherapeutic agent for treating AML.

Example 6 Apoptosis of Human Primary AML Cells by DP Agonists(Endogenous and Exogenous) and DP Antagonists

Referring to the results shown in FIG. 16, these experiments wereperformed with primary AML stem cells isolated from a patient. Theseresults strongly support the fact that Δ¹²-PGJ₃ (and other DP agonists)are effective even in human primary leukemia stem cells even from an AMLpatient. Human primary AML cells isolated from the bone marrow of an AMLpatient (72% of the cells were CD133+) were treated in-vitro withvarious concentrations of Δ¹²-PGJ₃ (5, 50, 100 nM) in the presence orabsence of DP antagonists (CAY10471 and MK0524, both 10 nM) for 6 and 24h. In an identical experiment, the cells were also treated with asynthetic DP agonist, ZK118182 (100 nM). Apoptosis of cells (by annexinV staining) was measured using flow cytometry. These results are inagreement with the data described above with mouse AML stem cells, whichfurther supports the use of DP agonists as a therapy for leukemias.

Other Embodiments

Any improvement may be made in part or all of the compositions, kits,and method steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. Any statement herein as to the nature or benefits ofthe invention or of the preferred embodiments is not intended to belimiting, and the appended claims should not be deemed to be limited bysuch statements. More generally, no language in the specification shouldbe construed as indicating any non-claimed element as being essential tothe practice of the invention. This invention includes all modificationsand equivalents of the subject matter recited in the claims appendedhereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contraindicated by context.

What is claimed is:
 1. A method of treating leukemia in a subject,comprising administering to the subject a composition comprising atherapeutically effective amount of an agonist for a prostaglandin Dreceptor, the prostaglandin D receptor selected from the groupconsisting of DP1 and DP2/CRTH2.
 2. The method of treating leukemia ofclaim 1, further comprising inhibiting growth of a leukemia stem cell inthe subject.
 3. The method of treating leukemia of claim 2, wherein theinhibiting growth of a leukemia stem cell in the subject comprisesinducing apoptosis in the leukemia stem cell.
 4. The method of treatingleukemia of claim 3, wherein the leukemia stem cell is selected from thegroup consisting of an acute lymphocytic leukemia stem cell, an acutemyeloid leukemia stem cell, a chronic lymphocytic leukemia stem cell,and a chronic myeloid leukemia stem cell.
 5. The method of treatingleukemia of claim 3, wherein the leukemia stem cell is selected from thegroup consisting of an acute myeloid leukemia stem cell and a chronicmyeloid leukemia stem cell.
 6. The method of treating leukemia of claim1, wherein the DP agonist is a cyclopentenone prostaglandin.
 7. Themethod of treating leukemia of claim 6, wherein the cyclopentenoneprostaglandin comprises an alkylidenecyclopentenone structure.
 8. Themethod of treating leukemia of claim 1, wherein the DP agonist isΔ¹²-prostaglandin J₃(Δ¹²-PGJ₃).
 9. The method of treating leukemia ofclaim 1, wherein the DP agonist is9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid, methyl ester(PGD₂ME).
 10. The method of treating leukemia of claim 1, wherein the DPagonist is[[4-[5R-chloro-2Z-[3R-cyclohexyl-3S-hydroxy-1R-propenyl]-3S-hydroxycyclopentyl]-2R-butenyl]oxy]-acetic acid, isopropyl ester (ZK118182).
 11. The method oftreating leukemia of claim 1, wherein the DP agonist isΔ¹²-prostaglandin J₂ (Δ¹²-PGJ₂).
 12. The method of treating leukemia ofclaim 1, wherein the DP agonist is 15-deoxy-Δ^(12,14)-PGJ₂.
 13. Themethod of treating leukemia of claim 1, wherein the DP agonist is16,16-dimethyl-Δ¹²-PGJ₃.
 14. The method of treating leukemia of claim 1,wherein the composition comprising a therapeutically effective amount ofa prostaglandin D receptor (DP) agonist further comprises an anti-cancerdrug.
 15. The method of treating leukemia of claim 14, wherein theanti-cancer drug is selected from the group consisting of imatinib,nilotinib, dasatinib, cytarabine, and doxorubicin.
 16. A pharmaceuticalformulation comprising a prostaglandin D receptor (DP) agonist and apharmaceutically acceptable carrier.
 17. The pharmaceutical formulationof claim 16 further comprising an anti-cancer drug.
 18. Thepharmaceutical formulation of claim 16, wherein the DP agonist is acyclopentenone prostaglandin.
 19. The pharmaceutical formulation ofclaim 18, wherein the cyclopentone prostaglandin comprises analkylidenecyclopentenone structure.
 20. The pharmaceutical formulationof claim 16, wherein the DP agonist is selected from the groupconsisting of Δ¹²-prostaglandin J₃ (Δ¹²-PGJ₃) or a derivative thereof;9α,15S-dihydroxy-11-oxo-prosta-5Z,13E-dien-1-oic acid, methyl ester(PGD₂ME);[[4-[5R-chloro-2Z-[3R-cyclohexyl-3S-hydroxy-1R-propenyl]-3S-hydroxycyclopentyl]-2R-butenyl]oxy]-aceticacid, isopropyl ester (ZK118182); Δ¹²-prostaglandin J₂ (Δ¹²-PGJ₂);15-deoxy-Δ^(12,14)-PGJ₂; and 16,16-dimethyl-Δ¹²-PGJ₃.
 21. Thepharmaceutical formulation of claim 17, wherein the DP agonist isΔ¹²-prostaglandin J₃(Δ¹²-PGJ₃) and the anti-cancer drug is imatinib.